Formulations of Human Parathyroid Hormone (PTH) and Methods for Producing Same

ABSTRACT

Stable liquid pharmaceutical formulations comprising human parathyroid hormone are provided herein. In various embodiments, the stable liquid pharmaceutical formulations comprise human parathyroid hormone (huPTH); a buffering agent to maintain the pH range of the solution from 3 to 6; one or more stabilizing agents selected from the group consisting of sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino acids; a tonicity modifier; water; and optionally a parenterally acceptable preservative; and wherein said solution is sterile and ready for parenteral administration to a human patient.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a U.S. National Phase Entry of InternationalPCT Application No. PCT/CN2020/082163 having an international filingdate of Mar. 30, 2020, which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing in the form of a“paper copy” (PDF File) and a file containing the referenced sequences(SEQ ID NO: 1-SEQ ID NO: 2) in computer readable form (ST25 format textfile) which is submitted herein. The Sequence Listing is shown usingstandard three letter code for amino acids, as defined in 37 C.F.R.1.822.

BACKGROUND OF THE INVENTION

Parathyroid hormone (PTH), also called parathormone or parathyrin, is ahormone secreted by the parathyroid glands that regulates the serumcalcium through its effects on bone, kidney, and intestine. PTHinfluences bone remodeling, which is an ongoing process in which bonetissue is alternately resorbed and rebuilt over time. PTH is secreted inresponse to low blood serum calcium levels. PTH indirectly stimulatesosteoclast activity within the bone matrix in order to release moreionic calcium into the blood to elevate a low serum calcium level.Disorders that yield too little or too much PTH, such ashypoparathyroidism, hyperparathyroidism, and paraneoplastic syndromescan cause bone disease, hypocalcemia, and hypercalcemia.

PTH is a polypeptide containing 84 amino acids, which is a prohormone.It has a molecular mass around 9500 Da. Studies in humans with certainforms of PTH have demonstrated an anabolic effect on bone and haveprompted significant interest in its use for the treatment ofosteoporosis and related bone disorders. Using the N-terminal 34 aminoacids of the bovine and human hormone, it has been demonstrated inhumans that parathyroid hormone enhances bone growth particularly whenadministered in pulsatile fashion by the subcutaneous route.Teriparatide (PTH 1-34) (FORTED®) is approved in the United States fortreatment of osteoporosis in those at high risk of fracture includingpostmenopausal women, men with primary or hypogonadal osteoporosis andmen and women with glucocorticoid-associated osteoporosis. A slightlydifferent form of PTH, human PTH (1-38) has shown similar results(Cusano N E et al., J Clin Endocrinol Metab., 98:137-144, 2013).

Unfortunately, like many small molecule therapeutics, PTH is susceptibleto degradation by proteases and labile due to degradation. In fact, itis more labile than the traditional small molecules. PTH is highlysensitive to deamidation, clips, aggregation and oxidation primarily atN-terminal amino acids, e.g., methionine residues in the positions 8 and18 giving rise to oxidized PTH species. Furthermore, it can getdeamidated at asparagine residue in position 16. There is a probabilityof truncation of polypeptide chain at N-terminal and C-terminals due tobreakage of peptide bond. All these reactions can significantly hamperthe bioactivity of this protein.

Improved formulations of PTH which serve to prevent these adversereactions are much needed.

Incorporation by Reference

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceuticallyuseful PTH formulation in the form of a stabilized liquid solutioncontaining a parathyroid hormone (PTH) in a therapeutically effectiveamount. The solution is storage stable and, in sterile form, may bestored in vials or cartridges ready for parenteral administration inhuman patients.

In various embodiments, the stable liquid formulations comprise humanparathyroid hormone (huPTH) in a concentration of about 100-2000 μg/ml;an acetate or citrate buffer to maintain the pH range of the solutionfrom 3 to 7; a stabilizing agent selected from the group consisting ofsugars, salts, surfactants, proteins, chaotropic agents, lipids, andamino acids; water; and optionally a parenteraly acceptablepreservative; wherein said solution is sterile and ready for parenteraladministration to a human patient.

In one embodiment, the stable liquid formulation comprises huPTH (1mg/mL), 10 mM sodium citrate, 50 mM L-Met, 10 mM EDTA, 100 mM NaCl,0.01% polysorbate 20, pH 5.0.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an SEC-HPLC chromatogram comparing a standard referencehuPTH sample (top) with a huPTH sample that had undergone forcedoxidation (bottom).

FIG. 2 depicts an RP-HPLC chromatogram comparing a standard referencehuPTH sample (top) with a huPTH sample that had undergone forcedoxidation (bottom).

FIG. 3 depicts an CEX-HPLC chromatogram comparing a standard referencehuPTH sample (top) with a huPTH sample that had undergone forcedoxidation (bottom).

FIG. 4 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 5 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 6 depicts an CEX-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 7 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 8 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 9 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 10 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 11 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 12 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 13 depicts an CEX-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 14 depicts an CEX-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 15 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 16 depicts an RP-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 17 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 18 depicts an SEC-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 19 depicts an CEX-HPLC chromatogram comparing various huPTHformulations at 25° C. (top) and 37° C. (bottom).

FIG. 20 depicts an CEX-HPLC chromatogram comparing various huPTHformulations at 4° C.

FIG. 21 depicts an RP-HPLC chromatogram evaluating the effects of Tween20 vs Tween 80 on huPTH formulations at 37° C.

FIG. 22 is a bar graph depicting the effects of Tween 20 vs Tween 80 onhuPTH formulations at 37° C. after 5 days.

FIGS. 23-25 are bar graphs depicting the stability of variousformulations at 4° C., 25° C. and 37° C. after one week.

FIGS. 26-28 are bar graphs depicting RP-HPLC evaluation of various huPTHformulations at 4° C., 25° C. and 37° C.

FIGS. 29-31 are bar graphs depicting CEX-HPLC evaluation of varioushuPTH formulations at 4° C., 25° C. and 37° C.

FIGS. 32-34 are bar graphs depicting SEC-HPLC evaluation of varioushuPTH formulations at 4° C., 25° C. and 37° C.

FIGS. 35-37 are bar graphs depicting RP-HPLC evaluation of various huPTHformulations at 4° C., 25° C. and 37° C.

FIGS. 38-40 are bar graphs depicting CEX-HPLC evaluation of varioushuPTH formulations at 4° C., 25° C. and 37° C.

FIGS. 41-43 are bar graphs depicting SEC-HPLC evaluation of varioushuPTH formulations at 4° C., 25° C. and 37° C.

DETAILED DESCRIPTION

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

As used herein, certain terms have the following defined meanings.

As used herein, the term “comprising” is intended to mean that theformulations and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to defineformulations and methods, shall mean excluding other elements of anyessential significance to the combination when used for the intendedpurpose. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants from the isolationand purification method and pharmaceutically acceptable carriers, suchas phosphate buffered saline, preservatives, and the like. “Consistingof” shall mean excluding more than trace elements of other ingredientsand substantial method steps for administering the formulations of thisinvention. Embodiments defined by each of these transition terms arewithin the scope of this invention.

As used herein, the term “aqueous pharmaceutical formulation” or “liquidpharmaceutical formulation” refers to a formulation of a therapeuticallyeffective amount of an active ingredient in water suitable foradministration to a patient.

In various embodiments the active ingredient of the pharmaceuticalformulation is a biologically active hPTH is selected from the groupcomprising hPTH (1-34), hPTH (1-37), hPTH (1-38), hPTH (1-41) and hPTH(1-84). In various embodiments, the liquid formulation may incorporatethe full length, 84 amino acid form of parathyroid hormone, particularlythe human form, hPTH (1-84) (SEQ ID NO: 1),

(SEQ ID NO: 1) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

obtained either recombinantly, by peptide synthesis, or by extractionfrom human fluid (see, e.g. U.S. Pat. No. 5,208,041, incorporated hereinby reference).

In various embodiments, the PTH fragments incorporate at least the first34 N-terminal residues, such as PTH (1-37), PTH (1-38), PTH (1-41) andPTH (1-34) (SEQ ID NO: 2),

(SEQ ID NO: 2)   SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

Alternatives in the form of PTH variants incorporate from 1 to 5 aminoacid substitutions that improve PTH stability and half-life, such as thereplacement of methionine residues at positions 8 and/or 18 with leucineor other hydrophobic amino acid that improves PTH stability againstoxidation and the replacement of amino acids in the 25-27 region withtrypsin-insensitive amino acids such as histidine or other amino acidthat improves PTH stability against protease. These forms of PTH areembraced by the term “parathyroid hormone” as used generically herein.The hormones may be obtained by known recombinant or synthetic methods,such as described in U.S. Pat. No. 4,086,196, incorporated herein byreference.

In various embodiments, the pharmaceutical formulation typicallycontains about 0.01 mg/mL to about 5 mg/mL of PTH, about 0.1 mg/mL toabout 2.5 mg/mL of PTH, or about 0.5 mg/mL to about 1 mg/mL of PTH. Invarious embodiments, the pharmaceutical formulation contains about 0.25mg/mL of PTH, about 0.5 mg/mL of PTH, about 1 mg/mL of PTH or about 2mg/mL of PTH. In various embodiments, the pharmaceutical formulationcontains 0.1 mg/mL to 1 mg/mL of PTH. In one embodiment, the formulationcontains 1 mg/mL of PTH.

The pharmaceutical formulations are generally formulated appropriatelyfor immediate use. In various embodiments when the pharmaceuticalformulation is not to be administered immediately, the PTH can beformulated in a formulation suitable for storage. One such formulationis a lyophilized formulation of the PTH together with a suitablestabilizer. Alternatively, the PTH can be formulated for storage in asolution with one or more suitable stabilizers. Any such stabilizerknown to one of skill in the art without limitation can be used. Invarious embodiments, stabilizers suitable for lyophilized preparationsinclude, but are not limited to, sugars, salts, surfactants, proteins,chaotropic agents, lipids, and amino acids. In various embodiments,stabilizers suitable for liquid preparations include, but are notlimited to, sugars, salts, surfactants, proteins, chaotropic agents,lipids, and amino acids.

As used herein, the term “buffer” or “buffer solution” refers to agenerally aqueous solution comprising a mixture of an acid (usually aweak acid, e.g. acetic acid, citric acid, imidazolium form of histidine)and its conjugate base (e.g. an acetate or citrate salt, for example,sodium acetate, sodium citrate, or histidine) or alternatively a mixtureof a base (usually a weak base, e.g. histidine) and its conjugate acid(e.g. protonated histidine salt). The pH of a “buffer solution” willchange very only slightly upon addition of a small quantity of strongacid or base due to the “buffering effect” imparted by the “bufferingagent”. The phrase “buffer system” means a mixture containing at leasttwo buffers. In various embodiments of the invention, the bufferingagent incorporated into the pharmaceutical formulation includes any acidor salt combination which is pharmaceutically acceptable and capable ofmaintaining the aqueous solution at a pH range of 3 to 7. In variousembodiments, the buffer maintains a pH of about 3.0 to about 7.0. Invarious embodiments, the buffer maintains a pH of about 3.0, a pH ofabout 4.0, a pH of about 5.0, a pH of about 6.0, or a pH of about 7.0.

Any buffer that is capable of maintaining the pH of the formulation atany pH or within any pH range provided above is suitable for use in thepharmaceutical formulations of the present disclosure, provided that itdoes not react with other components of the formulation, cause visibleprecipitates to form, or otherwise cause the active ingredient to becomechemically destabilized. Examples of suitable buffers are well known inthe literature (see, for example, Allen Jr, Loyd V, ed. (2012)Remington: The Science and Practice of Pharmacy, 22^(nd) ed.,Pharmaceutical Press). In various embodiments, the buffer used in thepharmaceutical formulation comprises a component selected from the groupconsisting of succinate, citrate, malate, edentate, histidine, acetate,adipate, aconitate, ascorbate, benzoate, carbonate, bicarbonate,maleate, glutamate, lactate, phosphate, and tartarate, or a mixture ofthese buffers.

The concentration of the buffer is selected so that pH stabilization aswell as sufficient buffering capacity is provided. In variousembodiments, buffer systems are acetate or citrate sources. In variousembodiments, the buffer system is a citrate source. In variousembodiments, the buffer is present at about 5 mM, at about 10 mM, atabout 15 mM or about 20 mM. In other embodiments, the buffer is presentin the formulation at a concentration of from 0.5 to 100 mM, from 0.75to 50 mM, from 1 to 20 mM, or from 10 to 20 mM. In one embodiment, thebuffer is present at about 10 mM. In one embodiment, the buffer iscitrate present at 10 mM.

In various embodiments of the invention, the stabilizing agentincorporated in the pharmaceutical formulation is selected from thegroup consisting of: sugars, salts, surfactants, proteins, chaotropicagents, lipids, and amino acids. In various embodiments, the stabilizingagent is selected from the group consisting of a polyol which includes asaccharide, preferably a monosaccharide or disaccharide, e.g., mannitol,glycine, glycerol, sorbitol or inositol and a polyhydric alcohol such asglycerine or propylene glycol or mixtures thereof; a chelator selectedfrom the group of EDTA, DTPA or EGTA; an amino acid(s) selected from thegroup of proline, alanine, arginine, asparagines, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, serine, threonine,tryptophan, tyrosine, and valine. Examples of suitable stabilizingagents have been described extensively in the art (see, for example,Allen Jr, Loyd V, ed. (2012) Remington: The Science and Practice ofPharmacy, 22^(nd) ed., Pharmaceutical Press).

In various embodiments of the invention, the stabilizing agentincorporated into the pharmaceutical formulation is a surfactant. A“surfactant” as used herein refers to an amphiphilic compound, i.e. acompound containing both hydrophobic groups and hydrophilic groups whichlowers the surface tension (or interfacial tension) between two liquidsor between a liquid and a solid. A “non-ionic surfactant” has no chargedgroups in its head. Examples of “non-ionic surfactants” include e.g.polyoxyethylene glycol alkyl ethers, such as octaethylene glycolmonododecyl ether, pentaethylene glycol monododecyl ether;polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such asdecyl glucoside, lauryl glucoside, octyl glucoside; polyoxyethyleneglycol octylphenol ethers, such as triton X-100; polyoxyethylene glycolalkylphenol ethers, such as nonoxynol-9; glycerol alkyl esters, such asglyceryl laurate; polyoxyethylene glycol sorbitan alkyl esters, such aspolysorbate; sorbitan alkyl esters, such as spans; cocamide MEA,cocamide DEA, dodecyldimethylamine oxide; block copolymers ofpolyethylene glycol and polypropylene glycol, such as poloxamers; andpolyethoxylated tallow amine (POEA). In various embodiments, thepharmaceutical formulations of the present invention can contain one ormore of these surfactants in combination. In various embodiments, thenon-ionic surfactants for use in the pharmaceutical formulations areselected from the group consisting of polysorbates such as polysorbate20, 40, 60 or 80, and the concentration of the non-ionic surfactant isin the range of 0.01 to 0.08% (w/v), 0.015 to 0.06% (w/v), or 0.02 to0.04% (w/v), relative to the total volume of the formulation. In oneembodiment, the non-ionic surfactant is polysorbate 20 (i.e. Tween 20)with a concentration of 0.01% (w/v), relative to the total volume of theformulation.

In various embodiments of the invention, the chelator incorporated intothe stable liquid formulation is selected from the group consisting ofEDTA, DTPA or EGTA. In various embodiments, the chelator is EDTA at aconcentration of 10 mM.

In various embodiments of the invention, the amino acid incorporatedinto the stable liquid formulation is selected from the group consistingof proline, alanine, arginine, asparagines, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, serine, threonine, tryptophan,tyrosine, and valine. In various embodiments, the amino acid isL-methionine at a concentration of 50 mM.

In various embodiments of the invention, the formulation of the presentdisclosure contains a physiologically acceptable tonicity modifier. Thephrase “tonicity modifier” means a pharmaceutically acceptable inertsubstance that can be added to the formulation to adjust the tonicity ofthe formulation. Tonicity modifiers suitable for this invention include,but are not limited to, sodium chloride, potassium chloride, mannitol,sucrose, dextrose, sorbitol, glycerin and other pharmaceuticallyacceptable tonicity modifier. When a tonicity agent is present, it ispreferably present in an amount sufficient to make the liquidformulation approximately isotonic with bodily fluids (i.e., about 270to about 300 mOsm/L) and suitable for parenteral injection into amammal, such as a human subject, into dermal, subcutaneous, orintramuscular tissues or IV. Isotonicity can be measured by, forexample, using a vapor pressure or ice-freezing type osmometer.

In various embodiments of the invention, the pharmaceutical formulationwill contain a tonicity modifier selected from the group consisting ofpotassium chloride, calcium chloride, sodium chloride, sodium phosphate,potassium phosphate and sodium bicarbonate, and the concentration willbe in the range of 10 to 200 mM, 20 to 150 mM, or 30 to 100 mM. In oneembodiment, the tonicity modifier is sodium chloride at a concentrationof 100 mM.

In various embodiments of the invention, the stable liquid formulationmay also include a parenterally acceptable preservative selected fromthe group consisting of cresols, benzyl alcohol, phenol, benzalkoniumchloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol,methyl paraben, propyl paraben, thimerosal and phenylmercuric nitrateand acetate.

As used herein, the terms “sterile water” or “water for injection” referto a sterile, nonpyrogenic preparation of water for injection whichcontains no bacteriostat, antimicrobial agent or added buffer. Ingeneral, the osmolar concentration of additives totals at least 112mOsmol/liter (two-fifths of the normal osmolarity of the extracellularfluid −280 mOsmol/liter).

The pharmaceutical formulations of the present invention are suitablefor parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical formulation includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical formulation through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical formulation by injection of theformulation, by application of the formulation through a surgicalincision, by application of the formulation through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous injection, intraperitoneal injection, intramuscularinjection, intrasternal injection, intravenous injection, intraarterialinjection, intrathecal injection, intraventricular injection,intraurethral injection, intracranial injection, intrasynovial injectionor infusions; or kidney dialytic infusion techniques. The presentinvention is related to a stable aqueous pharmaceutical formulation foruse in a pre-filled syringe, vial, cartridge, or pen.

The pharmaceutical formulations are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration. Preferably, a therapeutically effective dose of thepolypeptide formulations described herein will provide therapeuticbenefit without causing substantial toxicity.

In one embodiment, the stable liquid formulation comprises huPTH (1mg/mL), 10 mM sodium citrate, 50 mM L-Met, 10 mM EDTA, 100 mM NaCl,0.01% polysorbate 20, pH 5.0

The examples which follow are illustrative of the invention and are notintended to be limiting.

EXAMPLE 1 Formulation Stability Method Development

In the development of a method for evaluating stability, a testformulation of huPTH (1 mg/mL PTH) underwent forced oxidation with 0.01%H₂O₂ and was then subjected to SEC-HPLC, RP-HPLC, and CEX-HPLC asdescribed in the Additional Methods section below.

The results of the SEC-HPLC analysis is depicted in FIG. 1 . As depictedin FIG.1, the SEC main peak shifts to an earlier elution time,indicating aggregation caused by forced oxidation. The results of theRP-HPLC analysis is depicted in FIG. 2 . As depicted in FIG. 2 , the RPmain peak splits into four eluting peaks, indicating more hydrophilicspecies of PTH caused by forced oxidation. The results of the CEX-HPLCanalysis is depicted in FIG. 3 . As depicted in FIG. 3 , the main peakshifts to an earlier elution time, indicating less positively chargedspecies such as deamindated species caused by forced oxidation.

EXAMPLE 2 Screening Studies of pH and Buffers

This example demonstrates the effects of pH and varying bufferconditions on the stability of liquid huPTH formulations. RecombinanthuPTH (1 mg/mL) having the amino acid sequence set forth in SEQ ID NO: 2was used as an active ingredient in the formulations. Table 1 provides asummary of the formulations that were tested in this example.

TABLE 1 10 mM sodium acetate, 140 mM NaCl, pH 4.0 10 mM sodium acetate,140 mM NaCl, pH 5.0 10 mM citrate, 140 mM NaCl, pH 5.0 10 mM sodiumphosphate, 140 mM NaCl, pH 6.0 10 mM histidine, 140 mM NaCl, pH 6.0 10mM sodium phosphate, 140 mM NaCl, pH 7.0 10 mM Tris, 140 mM NaCl, pH 7.010 mM Tris, 140 mM NaCl, pH 8.0 10 mM glycine, 140 mM NaCl, pH 9.0

The results of SEC-HPLC, RP-HPLC and CEX-HPLC performed at 25° C. and37° C. are depicted in FIGS. 4-6 . The results of SEC-HPLC and RP-HPLCperformed at 4° C. are depicted in FIGS. 7-8 . These acceleratedstability studies at 25° C. and 37° C. show that huPTH may have beststability profile at pH 5.

EXAMPLE 3 Screening Study for Excipients and Surfactants at Optimized pHRange

This example demonstrates the effects of various excipients andsurfactants on the stability of liquid huPTH formulations at pH 5.0.Recombinant huPTH (1 mg/mL) having the amino acid sequence set forth inSEQ ID NO: 2 was used as an active ingredient in the formulations. Thefixed formulation parameters were pH 5.0 and 1.0 mg/mL huPTH.

To screen the effects of various excipients, ten different formulationswere prepared and stored in type 1 glass vials with TFE stoppers.Stability analysis (RP-HPLC, CEX-HPLC and SEC-HPLC, pH, Osmolarity,UV-VIS) was performed at 4° C., 25° C., 37° C. at 0, 1, 2, 4, 8, 12weeks. Table 2 provides a summary of the formulations that were testedin this example.

TABLE 2 10 mM sodium citrate, 50 mM ZnCl₂, pH 5.0 10 mM sodium citrate,10 mM L-Met, 50 mM ZnCl₂, pH 5.0 10 mM sodium citrate, 10 mM L-Met, 5%(w/v) Manitol, pH 5.0 10 mM sodium citrate, 10 mM L-Met, 5% (w/v)Sorbitol, pH 5.0 10 mM sodium citrate, 10 mM L-Met, 9% (w/v) Sucrose, pH5.0 10 mM sodium citrate, 10 mM L-Met, 5% (w/v) Trehalose, pH 5.0 10 mMsodium citrate, 10 mM L-Met, 10% (v/v) Propylene Glycol, pH 5.0 10 mMsodium citrate, 10 mM L-Met, 10% (v/v) Glycerol, pH 5.0 10 mM sodiumcitrate, 10 mM L-Met, 10% (w/v) Kolliphor P188, pH 5.0 10 mM sodiumcitrate, 10 mM L-Met, 100 mM NaCl, pH 5.0

The results of SEC-HPLC, RP-HPLC and CEX-HPLC performed at 4° C., 25° C.and 37° C. are depicted in FIGS. 9-14 . Initial accelerated stabilitystudies at 25° C. and 37° C. show that stability of huPTH at pH 5 can befurther increased in the presence of certain formulation excipients.Using ZnCl₂ as a bench mark, three formulation excipients provide betterstability profile for huPTH in aqueous solutions and SEC-HPLC, RP-HPLCand CEX-HPLC data show that formulations #1, #2, #3, #5, #6, #9 and #10are most stable under stressed accelerated conditions.

To screen the effects of various surfactants, twelve differentformulations were prepared and stored in type 1 glass vials with TFEstoppers. Stability analysis (RP-HPLC, CEX-HPLC and SEC-HPLC, pH,Osmolarity, UV-VIS) was performed at 4° C., 25° C., 37° C. at 0, 1, 2,4, 8, 12 weeks. Table 3 provides a summary of the formulations that weretested in this example.

TABLE 3 10 mM sodium citrate, 10 mM L-Met, 50 mM ZnCl₂, pH 5.0, 0.01%(v/v) polysorbate 20 10 mM sodium citrate, 10 mM L-Met, 5% (w/v)Sorbitol, pH 5.0, 0.01% (v/v) polysorbate 20 10 mM sodium citrate, 10 mML-Met, 10% (v/v) Propylene Glycol, pH 5.0, 0.01% (v/v) polysorbate 20 10mM sodium citrate, 10 mM L-Met, 100 mM NaCl, pH 5.0, 0.01% (v/v)polysorbate 20 10 mM sodium citrate, 50 mM ZnCl₂, pH 5.0, 0.01% (v/v)polysorbate 20 10 mM sodium citrate, 10 mM L-Met, 50 mM ZnCl₂, pH 5.0,0.1% (v/v) polysorbate 20 10 mM sodium citrate, 10 mM L-Met, 5% (w/v)Sorbitol, pH 5.0, 0.1% (v/v) polysorbate 20 10 mM sodium citrate, 10 mML-Met, 5% (w/v) Sorbitol, pH 5.0, 0.5% (v/v) polysorbate 20 10 mM sodiumcitrate, 10 mM L-Met, 10% (v/v) Propylene Glycol, pH 5.0, 0.1% (v/v)polysorbate 20 10 mM sodium citrate, 10 mM L-Met, 100 mM NaCl, pH 5.0,0.1% (v/v) polysorbate 20 10 mM sodium citrate, 50 mM ZnCl₂, pH 5.0,0.1% (v/v) polysorbate 20 10 mM sodium citrate, 10 mM L-Met, 100 mMNaCl, pH 5.0, 0.1% (v/v) Kolliphor P188

The results of SEC-HPLC, RP-HPLC and CEX-HPLC performed at 4° C., 25° C.and 37° C. are depicted in FIGS. 15-20 . RP-HPLC data (25° C.) show thatformulations #1, #3, #4, #5, #6, #7, #9, #10, and #11 are best understressed accelerated conditions whereas formulations #2, #8 and #12 arenot stable. SEC-HPLC data (25° C.) show that all formulations except #8and #12 are stable. CEX-HPLC data show that all formulations except #2,#8 and #12 are stable.

It was thus demonstrated that combination of L-Met, propylene glycol,and polysorbate-80 can provide good stability for huPTH at pH 5.0. Andwith the exception of formulations #2, #8 and #12, all formulations arestable as of two weeks. This L-Met effect on huPTH stability is a novelfinding.

EXAMPLE 4 Effect of Tween 20 and Tween 80 on the stability of huPTH

This example evaluated the effects of Tween 20 (T20) or Tween 80 (T80)on the stability of various formulations tested in Examples 1-3. Table 4provides a summary of the formulations that were initially tested inthis example.

TABLE 4 0.01% T20, 150 mM NaCl, 10 mM NaOAC, pH 5.0 0.1% T20, 150 mMNaCl, 10 mM NaOAC, pH 5.0 0.01% T80, 150 mM NaCl, 10 mM NaOAC, pH 5.00.1% T80, 150 mM NaCl, 10 mM NaOAC, pH 5.0

The results of RP-HPLC performed at 37° C. is depicted in FIG. 21 . Thestability of the formulations at 37° C. after 5 days is depicted in FIG.22 . The results demonstrated that Tween 80 caused more degradation inPTH than Tween 20. In fact, 0.01% (v/v) Tween 80 caused more degradationthan 0.1% (v/v) Tween 20. The observed degradations are primarilyoxidations.

In a second study, the formulations listed in Table 5 were evaluated.

TABLE 5 150 mM NaCl, 10 mM methionine, 10 mM NaOAc, 0.01% Tween 20 150mM NaCl, 10 mM methionine, 10 mM NaOAc, 0.01% Tween 80 100 mM NaCl, 50mM methionine, 10 mM NaOAc, 0.01% Tween 20 100 mM NaCl, 50 mMmethionine, 10 mM NaOAc, 0.01% Tween 80 50 mM NaCl, 100 mM methionine,10 mM NaOAc, 0.01% Tween 20 50 mM NaCl, 100 mM methionine, 10 mM NaOAc,0.01% Tween 80

The stability of the formulations at 4° C., 25° C. and 37° C. at 1 weekis depicted in FIGS. 23-25 . The results of RP-HPLC, CEX-HPLC andSEC-HPLC performed at 4° C., 25° C. and 37° C. are depicted in FIGS.26-34 . These results demonstrate that L-Met concentration needs to beincreased from 10 mM to 50-100 mM in order to prevent Tween 80 inducedoxidation and the increase in the concentration of L-Met leads toprotective effect of huPTH stability and at above 50 mM, the protectiveeffect of L-Met appears to be maximized.

EXAMPLE 5 Evaluation of L-Met and EDTA Combinations on the Stability ofhuPTH

This example evaluated the effects of various combinations of L-Met andEDTA on the stability of huPTH. Table 6 provides a summary of theformulations that were initially tested in this example.

TABLE 6 10 mM sodium citrate, 50 mM L-Met, 5 mM EDTA, pH 5.0, 4%Sorbitol 0.01% polysorbate 20 10 mM sodium citrate, 50 mM L-Met, 5 mMEDTA, pH 5.0, 100 mM NaCl, 0.01% polysorbate 20 10 mM sodium citrate, 50mM L-Met, 10 mM EDTA, pH 5.0, 4% Sorbitol 0.01% polysorbate 20 10 mMsodium citrate, 50 mM L-Met, 10 mM EDTA, pH 5.0, 100 mM NaCl, 0.01%polysorbate 20 10 mM sodium citrate, 50 mM L-Met, 10 mM EDTA, pH 5.0,100 mM NaCl, 0.01% polysorbate 80 10 mM sodium citrate, 5 mM EDTA, pH5.0, 4% Sorbitol, 0.01% polysorbate 20 10 mM sodium citrate, 5 mM EDTA,pH 5.0, 100 mM NaCl, 0.01% polysorbate 20 10 mM sodium citrate, 10 mMEDTA, pH 5.0, 4% Sorbitol, 0.01% polysorbate 20 10 mM sodium citrate, 10mM EDTA, pH 5.0, 100 mM NaCl, 0.01% polysorbate 20 10 mM sodium citrate,150 mM L-Met, pH 5.0, 0.01% polysorbate 20 10 mM sodium citrate, 150 mML-Met, 5 mM EDTA, pH 5.0, 0.01% polysorbate 20

The results of RP-HPLC, CEX-HPLC and SEC-HPLC performed at 4° C., 25° C.and 37° C. are depicted in FIGS. 35-43 . These results demonstrate thatthe optimal formulation comprises 10 mM sodium citrate, 50 mM L-Met, 10mM EDTA, pH 5.0, 100 mM NaCl, 0.01% polysorbate 20. And, surprisingly,it appears that combination of sorbitol and EDTA can stabilize huPTH at25° C. Based on the findings described herein, potential processparameters can be as following: dilute poloysorbate 20 into formulationbuffer to make 10% (v/v) concentration; dialyze huPTH againstformulation buffer to make drug substance (DS); spike 10% (v/v) ofpolysorbate 20 into drug substance (DS) to make drug product (DP).

Additional Methods SEC-HPLC

-   SEC Column: Phenomenex Yarra 3 μm SECd200 LC Column, 300×4.6 mm,    s/no H15d152245-   Mobile phase: 0.3M NaCl, 50 mM NaH₂PO₄ pH 7.0-   Flow rate: 0.3 ml/min-   Detector: 220 nm-   Col. Temp: 25±3° C.-   Auto Sampler: 5±2° C.-   Injection: 20 μl of 1mg/ml protein

RP-HPLC

-   RP Column: Phenomenex Jupiter 5p C18 300A, Column 250×4.6 mm, s/no    172611-   Mobile phase A: 0.1% TFA, 99.9% ddH₂O-   Mobile phase B: 0.1% TFA, 99.9% ACN-   Flow rate: 0.5 ml/min-   Detector: 220 nm-   Col. Temp: 25±3° C.-   Auto-Sampler: 5±2° C.-   Injection: 20 μl of 1 mg/ml protein

CEX-HPLC

-   CEX Column: TOSOH Bioscience, LLC TSKgel SP-NPR Column, 4.6    ID×3.5cm, 2.5 μm, s/no-   K0054-81C-   Mobile phase A: 38.75 mM NaAc, 22.5% ACN pH 5.5-   Mobile phase B: 46.25 mM NaAc, 277.5 mM NaCl, 7.5% ACN pH 5.1-   Flow rate: 0.3 ml/min-   Detector: 220 nm-   Col. Temp: 25±3° C.-   Auto-Sampler: 5±2° C.-   Injection: 20 μl mg/ml protein

What we claim is:
 1. An aqueous pharmaceutical formulation comprising:(a) a therapeutically effective amount of a human parathyroid hormone(PTH); (b) a buffering agent to maintain the pH range of the solutionfrom 3 to 6; (c) an effective amount of one or more stabilizing agents;(d) a tonicity modifier; and (e) the balance being water, wherein saidsolution is sterile and ready for parenteral administration to a humanpatient.
 2. The formulation of claim 1, wherein the one or morestabilizing agents is selected from the group consisting of sugars,salts, surfactants, proteins, chaotropic agents, lipids, and aminoacids.
 3. The formulation of claim 1, wherein the one or morestabilizing agents is a polyol selected from the group consisting ofmannitol, glycerol, sorbitol, inositol and a polyhydric alcohols, andpropylene glycol or mixtures thereof.
 4. The formulation of claim 2,wherein the one or more stabilizing agents is a non-ionic surfactantselected from the group consisting of polysorbate 20, polysorbate 40,polysorbate 60 and polysorbate
 80. 5. The formulation of claim 2,wherein the one or more stabilizing agents is an amino acid selectedfrom the group consisting glycine, alanine, serine, aspartic acid,glutamic acid, threonine, tryptophan, lysine, hydroxy lysine, histidine,arginine, cystine, cysteine, methionine, phenylalanine, leucine,isoleucine amino acids and their derivatives.
 6. The formulation ofclaim 5, wherein the amino acid is L-methionine.
 7. The formulation ofclaim 1, wherein the one or more stabilizing agents is a chelatorselected from the group of EDTA, DTPA and EGTA.
 8. The formulation ofclaim 1, wherein the tonicity modifier is selected from the groupconsisting of sodium chloride, potassium chloride, mannitol, sucrose,dextrose, sorbitol, and glycerin.
 9. A formulation according to claim 1,wherein the human PTH comprises the amino acid sequence set forth in SEQID NO:
 1. 10. A formulation according to claim 1, wherein the human PTHcomprises the amino acid sequence set forth in SEQ ID NO:
 2. 11. Aformulation according to claim 1, wherein the formulation has a pH ofabout 4.5 to 5.5.
 12. The formulation according to claim 1, wherein thehuman PTH is present in the formulation at a concentration of 0.1 mg/mLto 10 mg/mL.
 13. The formulation according to claim 1, which furthercomprises a parenterally acceptable preservative.
 14. A stable aqueousformulation comprising 1 mg/mL human PTH, 10 mM sodium citrate, 50 mML-Met, 10 mM EDTA, 100 mM NaCl, 0.01% polysorbate 20, wherein theformulation has a pH of about 5.0.